Carrier for ligand immobilization

ABSTRACT

A carrier for ligand immobilization obtained by shrinking polysaccharide porous beads not less than 10% by a shrinkage rate defined by the following formula, and crosslinking the polysaccharide porous beads: Shrinkage rate (%)=(1−V 2 /V 1 )×100 (wherein, V 1  indicates the gel volume of polysaccharide porous beads before shrinkage, and V 2  indicates the gel volume of polysaccharide porous beads after shrinkage).

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/025,152, filed on Mar. 25, 2016, which is a National Stage Entry ofInternational Application No. PCT/JP14/75740, filed on Sep. 26, 2014,the disclosures of which are incorporated herein by reference in theirentireties. This application claims priority to Japanese PatentApplication No. 2013-211453 filed on Oct. 8, 2013, and Japanese PatentApplication No. 2013-202007, filed on Sep. 27, 2013, the disclosures ofwhich are incorporated herein by reference in their entireties.

TECHNICAL FIELD

In the first embodiment, the present invention relates to a process forproducing porous cellulose beads, and more specifically, to a processfor producing porous cellulose beads utilizing liquid-liquid dispersion.Also, in the second embodiment, the present invention relates to acarrier for ligand immobilization comprising polysaccharide porousbeads, and to an adsorbent using the same.

BACKGROUND ART (1) Regarding Porous Cellulose Beads (First Embodiment)

Cellulose is resistant to acid and basic solvents, and varioussubstituents can be added to cellulose by modifying the cellulose.Therefore, cellulose porous beads have been used as adsorbents forvarious substances (Patent Documents 1, 2).

Very few solvents can dissolve cellulose, and cellulose porous particlesare produced, generally by dissolving cellulose in a highly toxicsolvent such as calcium thiocyanate. However, for producing celluloseporous beads by the production method as described above, the handlingis difficult in terms of corrosiveness and safety, and it is the currentstate of art that providing equipment thereof is not easy.

Meanwhile, ion liquids, which are nonvolatile and have the property ofassuming liquid in a wide temperature range, attract attentions inrecent years. Ion liquids are mainly applied as functional solvents,solvents for an ionics device and tissue-derived biomaterials such aspolypeptide. Recently, ion liquids are found to dissolve cellulose, andare applied, for example, in production of fibers (Patent Document 3).However, ion liquids are expensive, and it is not easy to form cellulosein a low-cost method.

Under these circumstances, a process for forming cellulose beads in alow-cost and simple process has been reported (Patent Document 4).However, Patent Document 4 lacks description of a specific process forcontrolling the pore size, particle diameter and the like of cellulosebeads. Therefore, further improvement should be made for using it as anadsorbent or the like for various substances.

(1) Regarding Carrier for Ligand Immobilization (Second Embodiment)

As described above, porous cellulose beads have a room for improvementregarding the production process thereof. In addition, polysaccharideporous beads including such porous cellulose beads have a problem to besolved in production of a carrier for ligand immobilization using thesame. That is, polysaccharide porous beads are useful as a carrier forligand immobilization, and for example, Non-patent Document 1 teachesimmobilizing an affinity ligand to a carrier of porous cellulose beadsand using them as an adsorbent. The polysaccharide porous beads on whichligands are immobilized are generally packed in a column, and an objectto be treated is allowed to run through the column, and thus theobjective matter is adsorbed. However, when the strength of the carrierfor ligand immobilization is low, problems such as critical compressionof the carrier due to liquid feeding pressure, and increase in pressureloss arise. In particular, since the operation pressure increases withthe column scale and the linear velocity, increase in strength of acarrier for ligand immobilization is strongly demanded.

As a method for increasing the strength of a carrier for ligandimmobilization, a method of cross linking polysaccharide porous beads isknown. For example, Patent Document 5 increases the strength bycrosslinking a carrier of cellulose-based beads, and Patent Document 6increases the strength by crosslinking a carrier of agarose-based beads.

It goes without saying that a carrier for ligand immobilizationdesirably has excellent adsorption after immobilization of a ligand, aswell as having increased strength.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: JP-A-1-278534-   Patent Document 2: JP-A-11-158202-   Patent Document 3: JP-A-2008-248466-   Patent Document 4: WO2012/121258-   Patent Document 5: JP-A-2008-279366-   Patent Document 6: JP-W-2000-508361

Non-Patent Document

-   Non-patent Document 1: “Affinity Chromatography”, attributed to    Kenichi Kasai et al., Tokyo Kagaku Dozin, 1991

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

In the case of the first embodiment (improvement in porous cellulosebeads), it is an object of the present invention to provide a processfor producing porous cellulose beads that can be used as an adsorbentfor various substances stably and conveniently without using a highlytoxic solvent such as calcium thiocyanate, and controlling the beadcharacteristics of the cellulose beads.

In the case of the second embodiment (improvement in carrier for ligandimmobilization), it is an object of the present invention not only toimprove the strength but also to maintain and improve the adsorption bya ligand in using polysaccharide porous beads as a carrier for ligandimmobilization.

Solutions to the Problems 1. In the Case of the First Embodiment(Improvement in Porous Cellulose Beads)

Through diligent efforts in light of the problem of the first embodiment(improvement in porous cellulose beads), the inventors of the presentapplication found that the aforementioned problems are solved in themanner of the later-described invention (1), and eventually completedthe invention according to the first embodiment (improvement in porouscellulose beads). The later-described invention (2) and the followinginventions may be preferred.

(1) A process for producing porous cellulose beads, comprising the stepsof:

a) mixing an alkali aqueous solution and cellulose to prepare acellulose micro dispersion at low temperature,

b) adding water to the cellulose micro dispersion to prepare a celluloseslurry, and

d) bringing the cellulose slurry into contact with a coagulationsolvent.

(2) The process according to (1), further comprising the step of:

c) raising the temperature of the cellulose slurry, after the step b),

wherein the step d) is conducted after the step c).

(3) The process according to (1) or (2), wherein an alkali concentrationof the cellulose micro dispersion is not less than 8 wt % and not morethan 10 wt %.

(4) The process according to any one of (1) to (3), wherein an alkaliconcentration of the cellulose slurry is not less than 5 wt %.

(5) The process according to any one of (1) to (4), wherein thetemperature at which the cellulose slurry is prepared is not less than4° C. and not more than 20° C.

(6) The process according to any one of (1) to (5), wherein afterliquid-liquid dispersing the cellulose slurry in a water-insolubleliquid which is a dispersion medium to prepare droplet, theliquid-liquid dispersion is brought into contact with a coagulationsolvent.

(7) The process according to any one of (1) to (6), whereinconcentration of the cellulose in the cellulose slurry is 1-7 wt %.

(8) The process according to any one of (1) to (7), wherein thecellulose is either regenerated cellulose, crystalline cellulose,microcrystalline cellulose, or cellulose acetate.

(9) The process according to (8), wherein a degree of polymerization ofthe cellulose is not more than 1000.

(10) The process according to any one of (1) to (9), wherein thewater-insoluble liquid is dichlorobenzene, hexane, ethyl acetate,straight-chain saturated fatty acid having 6 to 12 carbons, unsaturatedfatty acid having 16 to 24 carbons, animal fats and vegetable oilshaving a melting point of not more than 100° C., hydrogenated animalfats and vegetable oils, fractionated oil prepared by fractionating andpurifying a high-melting point fraction of animal fats and vegetableoils or hydrogenated animal fats and vegetable oils, unsaturated fattyacid triglycerides, edible waxes, fats and oils from microalgae, fatsand oils from microorganisms, medium-chain fatty acid triglycerides, orunsaturated fatty acid triglycerides.

(11) The process according to any one of (1) to (9), wherein thecoagulation solvent contains alcohols or glycols.

(12) The process according to (11), wherein the alcohols is at least oneselected from the group consisting of isobutanol, 2-butanol,sec-butanol, 2-methyl-2-propanol, 1-propanol, 2-propanol, ethanol, andmethanol.

(13) The process according to (11), wherein the glycols is at least oneselected from the group consisting of glycerol, ethylene glycol, andpropylene glycol.

(14) Porous cellulose beads produced by the process according to any oneof (1) to (13), wherein the porous cellulose beads have an exclusionlimit molecular weight of 1.0×10⁶ to 1.0×10¹¹.

(15) The porous cellulose beads according to (14), wherein the porouscellulose beads have a median particle diameter of 50 μm to 100 μm.

2. In the Case of the Second Embodiment (Improvement in Carrier forLigand Immobilization)

Through diligent efforts for solving the problem of the secondembodiment, the inventors of the present application unexpectedly foundthat a specific strength improving means not only increases the strengthof the carrier, but also maintains and improves the adsorption by aligand. To be more specific, inventors found that by subjectingpolysaccharide porous beads to a shrinking treatment, the compressivestrength increases, and not only critical compression and pressure losscan be prevented, but also adsorption when a ligand is immobilized ismaintained, improved, preferably improved, and completed also theinvention according to the second embodiment (improvement in carrier forligand immobilization).

That is, the invention according to the second embodiment (improvementin carrier for ligand immobilization) is as follows.

(16) A carrier for ligand immobilization obtained by shrinkingpolysaccharide porous beads not less than 10% by a shrinkage ratedefined by the following formula, and crosslinking the polysaccharideporous beads:Shrinkage rate (%)=(1−V ₂ /V ₁)×100(wherein, V₁ indicates the gel volume of polysaccharide porous beadsbefore shrinkage, and V₂ indicates the gel volume of polysaccharideporous beads after shrinkage).

(17) The carrier according to (16), wherein the carrier is producedthrough a shrinking step of bringing the polysaccharide porous beadsinto contact with a water-soluble organic solvent and alkali water.

(18) The carrier according to (17), wherein the carrier is produced byshrinking and crosslinking in the presence of a crosslinking agent inthe shrinking step, or by conducting a crosslinking step of crosslinkingobtained shrunk beads after the shrinking step.

(19) The carrier according to (18), wherein an additional crosslinkingstep of brining the beads obtained by the said crosslinking step intocontact with a crosslinking agent and alkali water is conducted once ormore.

(20) The carrier according to any one of (16) to (19), wherein thepolysaccharides is cellulose or agarose

(21) The carrier according to any one of (17) to (20), wherein thewater-soluble organic solvent is at least one selected from the groupconsisting of an alcohol solvent, a sulfoxide solvent, an amide solvent,a ketone solvent and an ether solvent.

(22) The carrier according to any one of (19) to (21), wherein analcohol solvent is not used in the additional crosslinking step.

(23) An adsorbent obtained by immobilizing a ligand on the carrieraccording to any one of (16) to (22).

(24) The adsorbent according to (23), wherein the ligand is an affinityligand.

(25) The adsorbent according to (24), wherein the affinity ligand isprotein A, protein G, or protein L.

(26) A method for purifying an antibody by affinity chromatography,comprising the steps of: bringing a source material into contact withthe adsorbent according to (24) or (25), to adsorb an antibody, andappropriately washing the antibody adsorbed on the adsorbent, and addingan eluent for liberating the antibody from the adsorbent, to collect theantibody from the eluate.

Specific examples recited in this description can be appropriately usedin combination of one or more kinds unless otherwise noted.

Effects of the Invention 1. In the Case of the First Embodiment(Improvement in Porous Cellulose Beads)

According to the process of the first embodiment of the presentinvention, by changing the solution temperature, and the concentrationof sodium hydroxide, it is possible to change the particle diameter andthe internal structure of the cellulose beads, and it becomes possibleto provide porous cellulose beads that can be used as an adsorbent forvarious substances.

2. In the Case of the Second Embodiment (Improvement in Carrier forLigand Immobilization)

In the second embodiment of the present invention, since thepolysaccharide porous beads are shrunk by a predetermined amount ormore, while they are crosslinked, it is possible to improve thecompressive strength. By immobilizing a ligand on the polysaccharideporous beads having improved compressive strength as a carrier, it ispossible to maintain or improve, preferably improve not only thestrength but also the adsorption.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a SEM photograph of a cellulose bead of the present invention(first embodiment) obtained in Example 1 regarding the first embodiment.

FIG. 2 is a SEM photograph of cellulose obtained in Comparative Example1 regarding the first embodiment.

FIG. 3 shows particle diameter distributions of cellulose beads obtainedin Example 1 to Example 4 regarding the first embodiment.

FIG. 4 shows K_(av) values of cellulose beads obtained in Example 1 toExample 4 regarding the first embodiment.

FIG. 5 is a chart regarding the second embodiment, and is a graphshowing the relationship between the shrinkage rate in the shrinking andcrosslinking step and the 20% compressive stress of the shrunk andcross-linked beads.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in the order ofimprovement in porous cellulose beads (first embodiment), andimprovement in a carrier for ligand immobilization (second embodiment).

1. Improvement in Porous Cellulose Beads (First Embodiment)

(1) Cellulose Micro Dispersion

In process of the present invention (first embodiment), first, celluloseis mixed with an alkali aqueous solution to prepare a cellulose microdispersion at low temperature. Retaining at low temperature contributesto producing excellent porous cellulose beads. In the micro dispersionpreparing step, for example, cellulose is micro dispersed in an alkaliaqueous solution of an alkali concentration of not more than 10 wt % andnot less than 7.5 wt % (preferably not less than 8 wt %, particularly9-8 wt %) at a temperature of about −5-10° C. (preferably 0° C.-4° C.).Alternatively, cellulose is mixed with an alkali aqueous solution sothat the mixture (micro dispersion) has the alkali concentration and thetemperature within the aforementioned ranges. Examples of the alkaliaqueous solution include a sodium hydroxide aqueous solution and apotassium hydroxide aqueous solution.

The concentration of cellulose in the cellulose micro dispersion is, forexample, not less than 5.5 wt %, and may be not less than 8 wt %. Theupper limit of the concentration of cellulose is for example, not morethan 20 wt %, preferably not more than 10 wt %.

As the cellulose, various celluloses can be used, and for example, anyof regenerated cellulose, crystalline cellulose, microcrystallinecellulose, and cellulose acetate may be used.

The degree of polymerization of cellulose is for example, not more than1000, preferably not more than 500, more preferably not more than 300.The lower limit of the degree of polymerization is, for example, notless than 10, preferably not less than 100, more preferably not lessthan 200.

The median particle diameter of the cellulose is, for example, not lessthan 10 μm, preferably not less than 20 μm, not less than 45 μm, and theupper limit thereof is, for example, not more than 500 μm, morepreferably not more than 300 μm, further preferably not more than 200μm.

(2) Cellulose Slurry

Preferably, after micro-dispersing cellulose in the alkali aqueoussolution, water is added, and the slurry temperature is raised. Thistemperature raising step is not necessary, and the cellulose slurry maybe prepared by adding water without changing the temperature. In anycase, when the micro dispersion is made into a slurry by addition ofwater, excellent porous cellulose beads are produced.

The alkali concentration of the cellulose slurry is, for example, notless than 3 wt %, preferably not less than 5 wt % and not more than 9 wt%, more preferably not less than 5 wt % and not more than 8 wt %(particularly not more than 7 wt %). In the present invention, althoughthe bead particle diameter can be made smaller by decreasing the alkaliconcentration, cellulose is no longer micro-dispersed when the alkaliconcentration is not more than 5 wt % (particularly less than 3 wt %).

The concentration of cellulose in the cellulose slurry is, for example,not less than 1 wt %, preferably not less than 2 wt %, and the upperlimit thereof is, for example, not more than 7 wt %, preferably not morethan 5.4 wt %.

In the cellulose slurry, the mass ratio between the amount of waterexisting in the micro dispersion (initial water amount), and the amountof adding water (water adding amount) is, for example, 95:5-30:70,preferably 90:10-40:60.

The higher the temperature of the cellulose slurry, the smaller theparticle diameter of beads can be made, and the higher the temperature,the larger the pore size of beads can be made. As a specific temperaturerange, the temperature of the cellulose slurry is preferably not lessthan 4° C. and not more than 25° C., further preferably not less than 4°C. and not more than 20° C. When the temperature of the cellulose slurryis not less than the upper limit, cellulose is no longermicro-dispersed.

When the micro dispersion is made into a slurry by raising thetemperature, the difference in temperature between the slurry and themicro dispersion is, for example, not less than 1° C., preferably notless than 5° C., more preferably not less than 10° C., and the upperlimit thereof is, for example, not more than 30° C., preferably not morethan 25° C., more preferably not more than 20° C.

(3) Droplet Preparing Step

Then, the cellulose slurry obtained in the manner as described above canbe brought into contact with a coagulation liquid to make porouscellulose beads, and as is necessary, the cellulose slurry may beprepared into droplet in which droplet (water phase) containingcellulose are dispersed in other liquid (oil phase) before it is broughtinto contact with the coagulation liquid. By coagulating cellulose afterformation of droplet, characteristics of porous cellulose beads arefurther improved. In the droplet preparing step, for forming celluloseporous beads, a cellulose slurry is dispersed in a water-insolubleliquid, and cellulose droplet are formed in the water-insoluble liquid.

As the water-insoluble liquid, halogenated hydrocarbons such asdichlorobenzene, aliphatic hydrocarbons such as hexane, esters(particularly acetic esters) such as ethyl acetate, straight-chainsaturated fatty acid having 6 to 12 carbons, unsaturated fatty acidhaving 16 to 24 carbons, animal fats and vegetable oils having a meltingpoint of not more than 100° C., hydrogenated animal fats and vegetableoils, fractionated oil prepared by fractionating and purifying ahigh-melting point fraction of animal fats and vegetable oils orhydrogenated animal fats and vegetable oils, unsaturated fatty acidtriglycerides, edible waxes, fats and oils from microalgae, fats andoils from microorganisms, medium-chain fatty acid triglycerides, andunsaturated fatty acid triglycerides can be recited. Thesewater-insoluble liquids can be used singly or in combination of two ormore kinds. Preferred water-insoluble liquids include halogenatedhydrocarbons, aliphatic hydrocarbons, esters, and particularlyhalogenated hydrocarbons.

The amount of water-insoluble liquid is not particularly limited as longas it can disperse the droplet phase of the cellulose slurry, and it is,for example, not less than 50 parts by mass, preferably not less than100 parts by mass, more preferably not less than 200 parts by mass,relative to 100 parts by mass of the cellulose slurry, and the upperlimit thereof is, for example, not more than 5000 parts by mass,preferably not more than 2000 parts by mass, more preferably not morethan 1000 parts by mass.

In preparing the droplet, a surfactant may be used as is necessary. Byusing a surfactant, it is possible to form the droplet phase of thecellulose slurry stably. As the surfactant, nonionic surfactants arepreferred, and specific examples include sorbitan fatty acid esters suchas sorbitan laurate, sorbitan stearate, sorbitan oleate, and sorbitantrioleate. These surfactants may be used singly or in combination of twoor more kinds. The amount of the surfactant is not particularly limited,and is for example, not less than 10 parts by mass, preferably not lessthan 30 parts by mass, more preferably not less than 50 parts by mass,relative to 100 parts by mass of cellulose, and the upper limit thereofis, for example, not more than 1000 parts by mass, preferably not morethan 300 parts by mass, more preferably not more than 200 parts by mass.

The surfactant may be, for example, added to a water-insoluble liquid tomake a mixture, and then the mixture may be brought into contact withthe cellulose slurry.

While the method for dispersing the cellulose slurry is not particularlylimited, the cellulose droplet can be uniformly dispersed in awater-insoluble liquid, for example, by stirring with a stirring bladeor by stirring with a homogenizer. Also a static mixer can be used. Whenthe stirring intensity is increased at this time, the size of thecellulose droplet decreases, and thus the particle diameter of theobtainable cellulose beads decreases.

The temperature during preparation of droplet can be set, for example,within the same range as the temperature range of the cellulose slurry.As long as this temperature range is maintained, the temperature may belowered or raised during preparation of droplet from the celluloseslurry, and the temperature difference between during production of thecellulose slurry and during production of droplet (temperature duringpreparation of droplet—temperature of the cellulose slurry) is typicallynot less than −5° C., preferably not less than −3° C., more preferablynot less than −1° C., and the upper limit thereof is, for example, notmore than 5° C., preferably not more than 3° C., more preferably notmore than 1° C., and most preferably the temperature difference is 0° C.

For adjusting the temperature during preparation of droplet within theaforementioned range, it is desired to preliminarily adjust thetemperature of the water-insoluble liquid (a surfactant may be containedas is necessary) before mixing with the cellulose slurry. In this case,the temperature difference between the cellulose slurry and thewater-insoluble liquid (temperature of water-insolubleliquid—temperature of cellulose slurry) is typically not less than −5°C., preferably not less than −3° C., more preferably not less than −1°C., and the upper limit thereof is, for example, not more than 5° C.,preferably not more than 3° C., more preferably not more than 1° C., andmost preferably the temperature difference is 0° C.

(4) Coagulating Step

The cellulose slurry or the cellulose droplet dispersion formed in thismanner is brought into contact with a liquid that can mingle with thecellulose slurry but shows cellulose coagulability, namely a coagulationsolvent. During mixing the dispersion and the cellulose coagulableliquid, cellulose droplet and the cellulose coagulable liquid come intocontact with each other, and cellulose beads are formed. Thereafter, theformed cellulose beads are collected.

It is recommended that mixing of the cellulose slurry or dropletdispersion with the coagulation solvent is conducted under a stirredconduction similar to that during preparation of the droplet. Thetemperature during this mixing (coagulation) is preferably comparable tothe temperature of the cellulose slurry or its droplet dispersion, andis for example, within ±10° C., preferably within ±5° C., morepreferably within ±2° C., relative to the temperature of the celluloseslurry or its droplet dispersion.

As the coagulation solvent, for example, alcohols, glycols and the likecan be used. Examples of the alcohols include isobutanol, 2-butanol,sec-butanol, 2-methyl-2-propanol (i.e., tert-butanol), 1-prop anol,2-prop anol, ethanol, and methanol, and these may be used singly or incombination of two or more kinds.

Examples of the glycols include glycerol, ethylene glycol, and propyleneglycol, and these may be used singly or in combination of two or morekinds.

(5) Isolating Step

The cellulose beads obtained by the first embodiment of the inventionare porous beads, and the size of the pore, and the particle diametercan be controlled by changing the temperature of cellulose slurry, andthe alkali concentration as described above.

The obtained porous beads can be isolated from the coagulation liquid bysolid-liquid separation by an appropriate method such as filtration,centrifugation or the like, and drying as necessary. In this isolatingoperation, porous cellulose beads may be washed with an appropriatesolvent (such as water, a water-soluble solvent such as alcohol).

The median particle diameter of the porous beads obtained in this manneris, for example, not less than 50 μm, preferably not less than 70 andthe upper limit thereof is, for example, not more than 100 μm, not morethan 95 μm.

The exclusion limit molecular weight of the porous cellulose beads is,for example, not less than 1.0×10⁶, preferably not less than 2.3×10⁶,more preferably not less than 1.0×10⁷, and the upper limit is, forexample, not more than 1.0×10¹¹, preferably not more than 8.0×10¹⁰.

(6) Other Respects

The porous cellulose beads obtained in the manner as described above canbe used as an adsorbent for various substances. They can be used also asa carrier for immobilizing a ligand. When they are used as a carrier, itis preferred that the porous cellulose beads are crosslinked. As amethod for crosslinking the porous cellulose beads, the inventionaccording to the second embodiment (crosslinking after shrinkage, orcrosslinking while shrinking, etc.) as will be described later may beconducted as it is, or a known crosslinking method may be conducted.

When a known crosslinking method is conducted, halohydrins such asepichlorohydrin, epibromohydrin, and dichlorohydrin; bifunctionalbisepoxides (bisoxirane); and multifunctional polyepoxides such asglycerol polyglycidyl ether (polyoxirane) can be recited as acrosslinking agent. Among others, the method shown in JP-A-2008-279366can be used particularly preferably. This publication is incorporated byreference in the present application.

The porous cellulose beads may be classified before crosslinking orafter crosslinking. The lower limit of particle diameter of porouscellulose beads after classification is, for example, 10 μm, preferably20 μm, more preferably 30 μm, and the upper limit is, for example, 200μm, preferably 150 μm, more preferably 125 μm.

Further, to the crosslinked carrier, a ligand may be immobilizedappropriately. The kind of the ligand and the immobilizing method can beappropriately selected from known scopes, and they may be conducted in asimilar manner as in the later-described second embodiment.

2. Improvement in Carrier for Ligand Immobilization (Second Embodiment)

Next, improvement in a carrier for ligand immobilization (secondembodiment) will be described.

(1) Polysaccharide Porous Beads

The carrier for ligand immobilization of the invention of the secondembodiment is crosslinked beads obtained by crosslinking polysaccharideporous beads. Polysaccharides used for polysaccharide porous beadsinclude agarose, cellulose, dextrin, chitosan, chitin, and derivativesthereof. Preferred polysaccharides include cellulose and agarose, withcellulose being particularly preferred.

The polysaccharide porous beads before crosslinking may be acommercially available product, or may be obtained by a known processusing polysaccharides. For example, the cellulose porous beads can beobtained by a process including granulation and coagulation afterdissolving or dispersing cellulose in an appropriate solution (forexample, JP-W-2009-242770, WO2996/025371, U.S. Pat. Nos. 4,634,470,5,410,034, WO2012/121258). Of course, the porous cellulose beads beforecrosslinking obtained by the first embodiment of the invention may beused.

The lower limit of the exclusion limit molecular weight ofpolysaccharide porous beads is, for example, 1.0×10⁵, preferably5.0×10⁵, more preferably 1.0×10⁶, and the upper limit is, for example,1.0×10¹², preferably 5.0×10¹¹, more preferably 1.0×10¹¹. By using thepolysaccharide porous beads having large exclusion limit molecularweight as described above, it is possible to obtain an adsorbent suitedfor separation of substances having large molecular weight likeantibodies.

The exclusion limit molecular weight can be determined in the followingmanner. Specifically, polysaccharide porous beads are packed in a column(packed beads capacity is defined as V_(t)), and a solution containingblue dextran 200, and various molecular weight markers is caused to runthrough the column. A liquid amount (V₀) required for the first peak tobe detected after starting running of blue dextran 200, and a liquidamount (V_(R)) required for the first peak to be detected after runningof each marker are determined, and a gel phase distribution coefficient(K_(av)) of each marker is determined according to the following formula(1). On a graph in which distribution coefficient (K_(av)) is assignedto the vertical axis, and a natural logarithm of molecular weight isassigned to the horizontal axis, the measurement result in each markeris plotted, and the following formula (2) is determined based on thepart showing linearity (in the formula, k and b are constants). In theformula (2), the molecular weight at which the distribution coefficient(K_(av)) is 0 is defined as exclusion limit molecular weight.K _(av)=(V _(R) −V ₀)/(V _(t) −V ₀)  (1)K _(av) =k×Ln(molecular weight)+b  (2)

(2) Shrinking Step

The second embodiment (improvement in carrier for ligand immobilization)of the present invention is featured in that polysaccharide porous beadsare shrunk. By utilizing shrinkage, it is possible to suitably increasethe compressive strength.

The degree of shrinkage can be evaluated based on the shrinkage ratedetermined by the following formula.Shrinkage rate (%)=(1−V ₂ /V ₁)×100

(wherein, V₁ indicates the gel volume of polysaccharide porous beadsbefore shrinkage, and V₂ indicates the gel volume of polysaccharideporous beads after shrinkage. In the present invention, as beingdescribed later, there is a case that beads are shrunk in the presenceof a crosslinking agent. In such a case, the V₁ indicates the gel volumeof polysaccharide porous beads before shrinking and crosslinking, and V₂indicates the gel volume of polysaccharide porous beads after shrinkingand crosslinking.)

In the present invention, the lower limit of the shrinkage rate is 10%,preferably 15%, more preferably 20%, and the upper limit is 60%,preferably 50%. The larger the shrinkage rate, the higher thecompressive strength (compressive stress) of the carrier can be made.

The gel volume means the volume of the sedimentation part when the beadssediment. Specifically, it is determined by using a sample slurryprepared by washing a beads-containing solution (reaction solution)before or after shrinkage, and replacing with RO water (Reverse Osmosiswater). The concentration of the sample slurry is roughly such that thegel volume concentration (gel volume/sample slurry volume) of the sampleslurry is 30-70% by volume, and a 50 mL centrifugal tube containing thesample slurry is fixed on a small vibrator (VIBRATORY PACKER, VP-4Davailable from SINFONIA TECHNOLOGY, or an equivalent article) at 25° C.,and the volume of the beads part is read from the scale of thecentrifugal tube after vibration is conducted until sedimentation ofbeads stops, and thus the gel volume in the sample slurry can bedetermined. Each of gel volumes V₁, V₂ in the above formula indicatesthe gel volume of the total amount of beads used in the shrinking step,and when the sample slurry is prepared by sampling part of the reactionsolution, the gel volumes V₁, V₉ of the total amount of beads aredetermined from the gel volume of the sample slurry and the sampledproportion.

For shrinking the polysaccharide porous beads by a predetermined amountor larger, it is recommended to bring the polysaccharide porous beadsinto contact with a water-soluble organic solvent, alkali and water. Bycontacting with alkali, shrinkage starts. Here, presence of thewater-soluble organic solvent results in significant increase in theshrinkage rate compared with the case where the water-soluble organicsolvent is absent.

Examples of the water-soluble organic solvent used in the secondembodiment include alcohol solvents such as methanol, ethanol, andpropanol; sulfoxide solvents such as dimethyl sulfoxide; amide solventssuch as dimethyl formamide, dimethyl acetamide, and N-methylpyrrolidone;ketone solvents such as acetone; ether solvents such as dioxane andtetrahydrofuran; and glycol solvents such as ethylene glycol anddiethylene glycol.

Preferred water-soluble organic solvents include alcohol solvents,sulfoxide solvents, amide solvents, ketone solvents, and ether solvents,and more preferred water-soluble organic solvents include methanol,ethanol, dimethyl sulfoxide, dimethyl formamide, dimethyl acetamide,acetone, and dioxane, and most preferred water-soluble organic solventsinclude ethanol and dimethyl sulfoxide. These may be used as two or morekinds of mixed solvent.

As alkali used for alkali water, an alkali metal-containing compound ispreferred, and for example, alkali metal hydroxide such as lithiumhydroxide, sodium hydroxide, and potassium hydroxide can be used. Thesealkalis may be used singly or in combination as appropriate. Preferredalkali is sodium hydroxide. While the alkali water is preferably aqueoussolution, it may be dispersion. It is usual to preliminarily preparealkali water, and add the alkali water to the reaction solution;however, the alkali water may be prepared in the reaction solution as isnecessary.

(3) Crosslinking

The present invention is also featured in that shrinking of thepolysaccharide porous beads is combined with crosslinking. Byimmobilizing a ligand on the polysaccharide porous beads having improvedstrength owing to improvement in the compressive strength by shrinkageand the compressive strength by crosslinking, as a carrier, it ispossible to maintain and improve the adsorption.

Specifically, in the shrinking step, it is preferred to cause shrinkingand crosslinking in the presence of a crosslinking agent (it ispreferred that the shrinking step is a shrinking and crosslinking stepthat also serves as a crosslinking step); however, the obtained shrunkbeads after the shrinking step may be crosslinked by a knowncrosslinking method. Also in crosslinking after the shrinking step, acrosslinking agent can be used. In any case, it is recommended that thedegree of crosslinking of the polysaccharide porous beads for use in theshrinking step is low (particularly, they are not cross-linked at all).As the degree of crosslinking of the polysaccharide porous beads beforethe shrinking step increases, shrinkage of the polysaccharide porousbeads at the shrinking step becomes more difficult, and a desiredshrinkage rate is difficult to be achieved.

As the crosslinking agent, halohydrins such as dichlorohydrin, or epoxycompounds can be preferably used, and examples of the epoxy compoundsinclude monoepoxy compounds such as epichlorohydrin and epibromohydrin;diepoxy compounds such as bisoxirane and diglycidyl ethers; andpolyepoxy compounds such as polyoxirane and polyglycidyl ethers.

(4) Shrinking Simplex Step, Shrinking and Crosslinking Step

When the shrinking step does not serve also as a crosslinking step(referred to as a shrinking simplex step), the step can be conducted bybringing the polysaccharide porous beads into contact with thewater-soluble organic solvent, alkali and water as described above. Whenthe shrinking step serves also as a crosslinking step (shrinking andcrosslinking step), the step can be conducted by bringing thepolysaccharide porous beads into contact with the water-soluble organicsolvent, the crosslinking agent, and the alkali water. The procedure,amounts, temperature and the like in bringing these polysaccharideporous beads, water-soluble organic solvent, alkali water, and thecrosslinking agent as necessary into contact with each other can beappropriately set. In the shrinking and crosslinking step, preferably, adispersion is prepared from the polysaccharide porous beads, thecrosslinking agent, and the water-soluble organic solvent, and thealkali aqueous solution is added to the dispersion. After allowing toreact for a predetermined time after adding the alkali aqueous solution,the alkali aqueous solution may be re-added once or more times. When thealkali aqueous solution is re-added, the crosslinking reaction beforere-addition is also referred to as a main shrinking and crosslinkingreaction.

The lower limit of the slurry concentration (concentration ofpolysaccharide porous beads) in the reaction solution in the shrinkingsimplex step, and the slurry concentration (concentration ofpolysaccharide porous beads) in the main shrinking and crosslinkingreaction (reaction before re-adding the alkali aqueous solution)solution in the shrinking and crosslinking step is, for example, 10% byvolume, preferably 20% by volume, more preferably 25% by volume, and theupper limit is, for example, 70% by volume, preferably 60% by volume,more preferably 50% by volume. The slurry concentration means gelvolume/total liquid amount (volume).

The lower limit of the rate of the water-soluble organic solvent in thereaction solution in the shrinking simplex step, and the rate of thewater-soluble organic solvent in the main shrinking and crosslinkingreaction (the reaction before re-adding the alkali aqueous solution)solution in the shrinking and crosslinking step is, for example, 0.30,preferably 0.40, more preferably 0.50, and the upper limit is, forexample, 0.90, preferably 0.80, more preferably 0.65. The rate of thewater-soluble organic solvent means organic solvent volume/(organicsolvent volume+alkali water volume), and the organic solvent volumeincludes the volume of the organic solvent used for gelation of thepolysaccharide porous beads. For example, when the water of aqueous gelof the polysaccharide porous beads is replaced with the organic solvent,the volume of the beads after replacement with the organic solvent isgenerally equivalent to the volume of the organic solvent.

The lower limit of the concentration of the crosslinking agent in themain shrinking and crosslinking reaction solution (volume ofcrosslinking agent/total liquid amount of reaction solution (volume))is, for example, 5% by volume, preferably 10% by volume, and the upperlimit is, for example, 50% by volume, preferably 40% by volume, morepreferably 30% by volume.

The lower limit of each of the alkali concentration in the reactionsolution in the shrinking simplex step, and the alkali concentration inthe main shrinking and crosslinking reaction solution in the shrinkingand crosslinking step is, for example, 0.1 M, preferably 0.3 M, morepreferably 0.5 M, and the upper limit is, for example, 2.0 M, preferably1.5 M, more preferably 1.2 M. In calculating the alkali concentration,the volume of the denominator indicates a sum of the organic solventvolume and the alkali water volume.

The lower limit of the temperature in causing both crosslinking andshrinking of the polysaccharide porous beads to proceed by adding thealkali water in the shrinking and crosslinking step is, for example, 0°C., preferably 20° C., more preferably 30° C., and the upper limit is,for example, 80° C., preferably 70° C., more preferably 50° C. Also inthe shrinking simplex step, the temperature ranges are applicable

(5) Additional Crosslinking Step

In the present invention, after crosslinking polysaccharide porous beadsby crosslinking at the time of shrinking reaction (shrinking andcrosslinking step) or by crosslinking after the shrinking reaction, anadditional crosslinking step for further crosslinking the obtainedcrosslinked beads may be conducted. The additional crosslinking step maybe conducted once or repeated several times. By conducting theadditional crosslinking step once or more, the compressive strength ofthe crosslinked beads can be further improved.

In the additional crosslinking step, there is a case that a shrinkingphenomenon little occurs even if the water-soluble organic solvent isused. The additional crosslinking step can be conducted in the samemanner as in the shrinking and crosslinking step described above exceptthat use of the water-soluble organic solvent is optional. Therefore,the specific examples of the crosslinking agent, and alkali water to beused, the procedure, and use amounts are also similar. The use amount ofthe water-soluble organic solvent in the aforementioned shrinking andcrosslinking step is applied while it is read as a preferred range inthe additional crosslinking step.

In the additional crosslinking step, it is preferred that an alcoholsolvent is not used. The preferred modes include the mode not using aprotonic organic solvent, and the mode not using a water-soluble organicsolvent, and also include the mode using an aprotic water-solubleorganic solvent as a water-soluble organic solvent. When the alcoholsolvent is not used, the crosslinking reaction is easy to proceed andthe strength can be further improved in comparison with the case wherethe alcohol solvent is used. As the aprotic water-soluble organicsolvent, aprotic solvents among the aforementioned water-soluble organicsolvents can be used singly or in combination.

(6) Preliminary Crosslinking Step

Further, in the present invention, prior to the shrinking step(including the shrinking and crosslinking step), a preliminarycrosslinking step for preliminarily crosslinking polysaccharide porousbeads may be conducted. The preliminary crosslinking step can beconducted by a similar operation as in the additional crosslinking stepexcept that the degree of cross linking (crosslinking agentconcentration or the like) is small. In the present invention, it ispreferred to conduct the shrinking step (including the shrinking andcrosslinking step) without conducting the preliminary crosslinking step.

In the preliminary crosslinking step, the shrinking and crosslinkingstep, crosslinking after the shrinking step, and the additionalcrosslinking step, a reaction promoter, a reducing agent such as sodiumborohydride or an inorganic salt may be used as necessary. By using theinorganic salt, it is possible to further increase the compressivestress of the crosslinked beads. Examples of the inorganic salt includehydrochlorides, sulfates, phosphates and borates of alkali metal oralkali earth metal, and sodium sulfate is particularly preferred. Theseinorganic salts may be used singly or in combination of two or morekinds. After end of crosslinking, a curing treatment may be conducted asis necessary. In the curing treatment, for example, a pressurizing andheating treatment using an autoclave is convenient.

(7) Crosslinked Beads (Carrier for Ligand Immobilization)

The crosslinked beads obtained in the manner as described above can beused as a carrier for ligand immobilization. The lower limit of the 20%compressive stress of the crosslinked beads is, for example, 0.06 MPa,preferably 0.072 MPa, more preferably 0.092 MPa, and the upper limit is,for example, 0.28 MPa, preferably 0.20 MPa. The 20% compressive stressmeans the stress required for compressing by 20% after causing thebeads-containing slurry to sediment on the filter having a pore diameterof 5.00 μm to such a degree that they no longer sediment even undervibration.

The crosslinked beads are desired to have higher linear velocity atwhich critical compression occurs. In the crosslinked beads of thepresent invention, the linear velocity of critical compression is, forexample, not less than 700 cm/h, preferably not less than 1000 cm/h,more preferably not less than 1500 cm/h. The linear velocity of criticalcompression means the linear velocity when water is run through a columnpacked with the crosslinked beads, and the inlet pressure continues torise to finally disable the liquid running.

The crosslinked beads may be classified as necessary by a screen or thelike. The lower limit of particle diameter of the crosslinked beadsafter classification is, for example, 10 μm, preferably 20 μm, morepreferably 30 μm, and the upper limit is, for example, 200 μm,preferably 150 μm, more preferably 125 μm.

(8) Ligand

By immobilizing a ligand on the crosslinked beads serving as a carrier,an adsorbent can be obtained. The crosslinked beads carrier of thepresent invention can maintain and improve the adsorption by the ligandfor its strength.

As the ligand, those having affinity with the object to be adsorbed canbe appropriately used, and examples of such a ligand include an affinityligand, a charged group, and a hydrophobic group, and these may beintroduced singly or may be introduced while a plurality of these arecombined appropriately. These ligands can be introduced by a knownmethod, and the obtained adsorbent can be suitably used as a columnpacking material for various chromatography such as affinitychromatography, ion exchange chromatography, chelate chromatography, andhydrophobic interaction chromatography. Further, the crosslinked beadscarrier of the present invention is suited for separation andpurification of an antibody as an objective substance because of itspore size, and the absorbent into which an affinity ligand, a chargedgroup, a hydrophobic group or the like can be suitably used forpurification of an antibody.

A preferred ligand in the present invention is an affinity ligand. Whilethe affinity ligand is not particularly limited as long as it has afeature of capable of specifically binding with an antibody or the likeas a target molecule, it is preferably a peptidic, proteinic orsynthetic compound. From the view point of specificity to a targetmolecule, a peptidic or proteinic ligand is further preferred, and amongothers, particularly preferred antibody affinity ligands include proteinA, protein G, protein L, protein H, protein D, protein Arp, proteinFcγR, antibody-binding synthetic peptide ligands and their relativesubstances. Protein used as a ligand in the present descriptionencompasses variants thereof. Natural products, genetically engineeredproducts and the like can be used without any restriction, and variousvariants that are generally produced can be used. Also those containingan antibody-binding domain and its variant, fusion proteins and so oncan also be used. For example, for improving the bindability with theantibody, those having a sequence modified so that the antibody bindingprotein is site-specifically immobilized to the substrate material (forexample, protein with controlled position and number of lysine residuesas described in JP 4179517 B1 and JP-A-2008-214350) can also be used.

Also usable is protein that is produced from a bacterial extract or aculture supernatant by combination and/or repetition of purifyingtechniques selected from the techniques such as molecular weightfractionation, fractionating precipitation method using variouschromatography including ion exchange chromatography, hydrophobicinteraction chromatography, gel filtration chromatography andhydroxyapatite chromatograph, and membrane separation technique.Preferred affinity ligands are protein A, protein G, protein L andvariants thereof, and protein A is particularly preferred. Protein Aattracts attentions as a ligand capable of specifically adsorbing andeluting immunoglobulin (IgG) or the like. An adsorbent on which proteinA is immobilized attracts attentions as a therapeutic adsorbent forrheumatism, hemophilia, and dilated cardiomyopathy. Also in the field ofantibody drug purification, an adsorbent capable of conductingpurification of antibodies such as IgG in large scale, at high speed, atlow cost is demanded.

Generally, protein A indicates one kind of cell wall protein produced bya gram positive bacterium, Staphylococcus aureus, and consists of asignal sequence S, five IgG binding domains (E domain, D domain, Adomain, B domain, C domain), and a XM region which is a cell wallbinding domain (attributed to Hober S. et al., “J. Chromatogr. B”, 2007,vol. 848, pp. 40-47). As protein A for use as a ligand for IgG affinitypurification, protein consisting exclusively of five IgG binding domains(SEQ ID NO: 1) is generally used. Protein A used herein also includesproteins having either one or more domains selected from E, D, A, B, andC domains, as well as the general protein A and protein consistingexclusively of E, D, A, B, and C domains, and it may be proteinconsisting exclusively of the one having, for example, a plurality of(e.g., three to eight, particularly five) C domains in series.

Preferred examples of protein A include protein A having alkaliresistance and orientation-controlled protein A. Since the carrier ofthe present invention has strengthened alkali resistance by the effectof crosslinking or the like, by strengthening the alkali resistance ofthe ligand, it is possible to increase the alkali resistance as anadsorbent. Examples of protein A having alkali resistance includeprotein in which an alkali sensitive residue (either one of asparagineresidue, glutamine residue and so on) in the protein A is deleted, orprotein in which the alkali sensitive residue is replaced with an alkaliresistant residue (for example, natural amino acid residues other thanan asparagine residue and a glutamine residue, preferably, natural aminoacid residues further excluding a cysteine residue, more preferably alysine residue, an asp artic acid residue, a leucine residue and so on)(for example, JP-W-2002-527107); protein having a domain (Z domain) inwhich the glycine residue at position 29 in B domain of protein A isreplaced with an alanine residue (for example, JP-B-8-11069); protein inwhich the glycine residue at position 29 in C domain of protein A isreplaced with a natural amino acid residue other than a glycine residue(for example, alanine residue, leucine residue, isoleucine residue,phenylalanine residue, tyrosine residue, tryptophan residue, glutamineresidue, arginine residue, or methionine residue), and protein in whicha plurality of (particularly five) C domains in which the residue atposition 29 is replaced are exclusively connected (for example,WO2010/110288).

Examples of orientation-controlled protein A include protein in which acysteine residue is granted (replaced, added, or the like) in the Cterminal or the N terminal of protein A (for example, JP-A-2008-101023);(A) protein in which not less than ½, preferably not less than ⅔, morepreferably not less than ¾, particularly preferably all of the lysineresidues in protein A are replaced with other amino acid residue, (B)preferably, protein wherein other amino acid is arginine, glutamine,asparagine, aspartic acid, glutamic acid, isoleucine, histidine orglycine in protein of (A), (C) more preferably, protein wherein one ormore lysine residues are granted (replaced, added or the like) in aterminal (particularly C terminal) in protein of (A) or (B) (forexample, WO2012/133349). Also orientation-controlled protein A havingalkali resistance by combining these modifications may be used.

The lower limit of the introducing amount of the affinity ligand is, forexample, 2 mg, preferably 4 mg, more preferably 10 mg per 1 mL ofcrosslinked beads, and the upper limit is, for example, 40 mg,preferably 30 mg, more preferably 20 mg per 1 mL of crosslinked beads.

The introducing amount of the affinity ligand can be determined by aknown method.

For immobilizing the affinity ligand on the crosslinked beads, variousknown immobilizing methods, e.g., a cyanogen bromide method, atrichlorotriazine method, an epoxy method, and a tresyl chloride methodcan be appropriately employed. For the reasons of safety, easiness ofimmobilizing reaction, and ability to immobilizing produced protein orpeptide with a relatively easy method, a method of introducing a formylgroup into crosslinked beads, and reacting the formyl group and an aminoacid of the affinity ligand (for example, WO2010/064437) is preferredtreatment.

Formyl groups can be introduced into the crosslinked beads by cuttingC—C bonds in a, 6-diol units contained in polysaccharides. When an epoxycompound such as epichlorohydrin is used as the crosslinking agent,formyl groups can also be introduced by oxidizing and cleaving diolsobtained by hydrolysis of epoxy groups. Regarding the amount of theformyl groups, the lower limit is, for example, 1 μmol, preferably 5μmol, more preferably 10 lima particularly preferably 20 μmol, mostpreferably 30 μmol per 1 mL of crosslinked beads, and the upper limitis, for example, 500 μmol, preferably 250 μmol, more preferably 125μmol, particularly preferably 60 μmol, most preferably 50 μmol, Activegroups remaining after introduction of the affinity ligand are subjectedto an inactivating

According to the present invention, since the strength of the carrier isstrengthened by compression, the adsorptive performance of the adsorbentin which the affinity ligand is immobilized is also maintained andimproved. When protein A is used as the affinity ligand, the adsorptivecharacteristic of the adsorbent can be evaluated, for example, by 5%dynamic binding capacity (DBC) of human immunoglobulin (IgG). When theadsorption treatment is conducted for a residence time (RT) of 3minutes, the lower limit of 5% DBC is, for example, 20 mg, preferably 30mg, more preferably 40 mg, per 1 mL of adsorbent, and the upper limitis, for example, 100 mg, preferably 60 mg.

The 5% DBC can be determined by allowing a phosphate buffer at pH 7.4 torun through the column packed with the adsorbent, and then allowing anIgG aqueous solution having a concentration of 1 mg/mL to run throughthe column.

The adsorbent can be used for purifying various target substances. Theadsorbent may be used while it is packed in a column as is necessary. Inone embodiment, the adsorbent is packed in a chromatography column. Thecolumn can be used for affinity chromatography by using a conventionalliquid chromatography device or the like.

Also, the present invention provides a method for purifying antibodiesfrom a mixture (source material). This method can include the step ofcontacting (loading) a mixture (source material) containing antibodiesin the condition that antibodies selectively bind with the adsorbentobtained in the present invention, and optionally the step of elutingantibodies from the adsorbent by applying (allowing to run through) aneluent (preferably elution buffer) prepared by changing at least onecondition (pH, salt strength). The antibodies can be collected from thiseluate.

As the elution buffer, more specifically, a salt having different pHfrom the pH at the time of bringing into contact with the mixturecontaining antibodies (loading solution), or a salt of higherconcentration can be used. This method can optionally include one ormore washing steps. The washing step can be conducted, for example,after antibodies have bound the adsorbent, and before the antibodies areeluted from the adsorbent. Adsorption and elution of antibodiesaccording to the present invention can be easily conducted in standardconditions as recommended for conventional commercially availableproducts, and for example, the website of GE healthcare can be referred,for example.

The purification method described above can be combined with otherchromatography to further improve the purification purity of antibodies.

The present application claims the benefit of priority based on thebenefits of priority based on Japanese patent application No.2013-202007 filed on Sep. 27, 2013 and Japanese patent application No.2013-211453 filed on Oct. 8, 2013. The entirety of each of descriptionsof Japanese patent application No. 2013-202007 filed on Sep. 27, 2013and Japanese patent application No. 2013-211453 filed on Oct. 8, 2013 isincorporated herein by reference.

EXAMPLES

Hereinafter, the present invention is described more specifically by wayof examples; however, it should be noted that the present invention isnot limited by the following examples, and the present invention can bepracticed with appropriate modification within the scope mentioned aboveor below, and any of such modification is included in the technicalscope of the present invention.

1. Improvement in Porous Cellulose Beads (First Embodiment) Example 1

8.65 g of powdery cellulose (pharmacopoeia cellulose PH-F20JP availablefrom Asahi Kasei Chemicals Corporation) was dispersed in 112 g of water,and retained at 4° C. Then, 35 g of 34.5 wt % sodium hydroxide(available from NACALAI TESQUE, INC.) aqueous solution was added andstirred to obtain a cellulose micro dispersion. After stirring for 20minutes, 16 g of water was added so that the concentration of sodiumhydroxide was 7.0 wt %, and the concentration of cellulose was 5.0 wt %,and the temperature was raised to 15° C., and thus a cellulose slurrywas obtained. In 890 g of 1,2-dichlorobenzene at 15° C. containing 9.2 gof sorbitan monooleate, the obtained cellulose slurry was dispersed toform a cellulose droplet dispersion. The cellulose droplet dispersionwas put into a cylindrical container having an inner diameter of 85 mm.A two-stage turbine blade having a blade diameter of 45 mm was used forstirring, and the interval of blades was set at 75 mm. The dispersionwas stirred at 600 rpm for 10 minutes, and 150 mL of methanol was addedto obtain cellulose in the form of beads. The temperature at the time offorming the beads was equivalent to the temperature at the time offorming the cellulose droplet dispersion. Regarding the particlediameter of the obtained beads, a median particle diameter determined byusing a laser diffraction/scattering particle diameter distributionmeasuring device (LA-950 available from HORIBA, Ltd.) was 90.2 μm. Theobtained cellulose beads were washed with methanol, and then washed withwater. Part of the collected cellulose beads were replaced with2-methyl-2-propanol, and freeze-dried, and then analyzed by a scanningelectron microscope (S-800 available from Hitachi, Ltd., hereinafterreferred to as SEM). As a result, it was confirmed that the cellulosebeads were porous beads as shown in FIG. 1.

The residual cellulose beads was classified by using a mesh with anopening of sieve of 38 μm and a mesh with an opening of sieve of 90 μmto collect beads within the range of 38 μm to 90 μm. The crosslinkingreaction was conducted in the later-described method. Gel distributioncoefficient (K_(av)) of the obtained crosslinked cellulose beads wascalculated by conducting column packing and measurement in thelater-described method. K_(av) value for the marker molecular weight of12400 was 0.723, K_(av) value for the marker molecular weight of 67000was 0.646, K_(av) value for the marker molecular weight of 115000 was0.595, and K_(av) value for the marker molecular weight of 440000 was0.525. The exclusion limit molecular weight calculated from these K_(av)values was 6.0×10¹⁰.

Example 2

Porous cellulose beads were obtained in the same manner and conditionsas in Example 1 except that the temperature of cellulose slurry wasraised to 10° C. and the temperature of 1,2-dichlorobenzene was 10° C.As a result, a particle median diameter of the obtained beads was 91.4μm. A crosslinking reaction was conducted in the same manner as inExample 1, and K_(av) was calculated. K_(av) value for the markermolecular weight of 12400 was 0.562, K_(av) value for the markermolecular weight of 67000 was 0.429, K_(av) value for the markermolecular weight of 115000 was 0.377, K_(av) value for the markermolecular weight of 440000 was 0.307, and K_(av) value for the markermolecular weight of 660000 was 0.261. The exclusion limit molecularweight calculated from these K_(av) values was 2.4×10⁷.

Example 3

Porous cellulose beads were obtained in the same manner and conditionsas in Example 1 except that the temperature of cellulose slurry wasraised to 20° C. and the temperature of 1,2-dichlorobenzene was 20° C.As a result, a particle median diameter of the obtained beads was 80.3μm. A crosslinking reaction was conducted in the same manner as inExample 1, and K_(av) was calculated. K_(av) value for the markermolecular weight of 12400 was 0.645, K_(av) value for the markermolecular weight of 67000 was 0.528, K_(av) value for the markermolecular weight of 115000 was 0.487, K_(av) value for the markermolecular weight of 440000 was 0.400, and K_(av) value for the markermolecular weight of 660000 was 0.374. The exclusion limit molecularweight calculated from these K_(av) values was 1.5×10⁸.

Example 4

Porous cellulose beads were obtained in the same manner and conditionsas in Example 1 except that the temperature of cellulose slurry and thetemperature of 1,2-dichlorobenzene were 4° C. As a result, a particlemedian diameter of the obtained beads was 96.3 μm. A crosslinkingreaction was conducted in the same manner as in Example 1, and K_(av)was calculated. K_(av) value for the marker molecular weight of 67000was 0.377, K_(av) value for the marker molecular weight of 115000 was0.335, K_(av) value for the marker molecular weight of 440000 was 0.179,and K_(av) value for the marker molecular weight of 660000 was 0.146.The exclusion limit molecular weight calculated from these K_(av) valueswas 2.4×10⁶.

FIG. 3 shows the particle diameter distributions in Example 1 to Example4. The bead diameter decreases as the beads formation temperature rises.By controlling the beads formation temperature, the particle diametercould be easily controlled. FIG. 4 shows K_(av) values in Example 1 toExample 4. The K_(av) value and the exclusion limit molecular weightcould be controlled by the beads formation temperature. The K_(av) valueindicates the diffusion behavior of the marker protein into beads. Thelarger the K_(av) value is, the more easily the protein diffuses intothe beads. The protein diffusion behavior reflects the pore size ofbeads, and a larger K_(av) value indicates a larger pore size.Therefore, Example 1 to Example 4 demonstrate that the pore size ofbeads can be controlled by the beads formation temperature.

Example 5

8.65 g of powdery cellulose was dispersed in 60 g of water, and retainedat 4° C. Then, 27 g of 31.9 wt % sodium hydroxide aqueous solution wasadded and stirred. Porous cellulose beads were obtained in the samemanner and conditions as in Example 4 except that after stirring for 20minutes, 77 g of water was added so that the concentration of sodiumhydroxide was 5.0 wt %, and concentration of cellulose was 5.0 wt %. Asa result, a particle median diameter of the obtained beads was 83.0 μm.

Comparative Example 1

Porous cellulose beads were obtained in the same manner and conditionsas in Example 1 except that 8.65 g of powdery cellulose was dispersed in128 g of water, and retained at 15° C., and then 35 g of 34.5 wt %sodium hydroxide aqueous solution was added so that the concentration ofsodium hydroxide was 7 wt %. As a result, the obtained cellulose was notin the shape of beads as shown in FIG. 2.

Comparative Example 2

8.65 g of powdery cellulose was dispersed in 112 g of water, andretained at 4° C., and then 51 g of 30. 3 wt % sodium hydroxide aqueoussolution was added so that the concentration of sodium hydroxide was 9wt %, and the concentration of cellulose was 5.0 wt %. The obtainedcellulose slurry at 4° C. was dispersed in 890 g of 1,2-dichlorobenzenecontaining 9.2 g of sorbitan monooleate to form a cellulose dropletdispersion. The cellulose droplet dispersion was put into a cylindricalcontainer having an inner diameter of 85 mm. A two-stage turbine bladehaving a blade diameter of 45 mm was used for stirring, and the intervalof blades was set at 75 mm. The dispersion was stirred at 600 rpm for 10minutes, and 150 mL of methanol was added to obtain cellulose in theform of beads. This method is the same condition as in WO2012-1212158.As a result, a particle median diameter of the obtained beads was 111.0μm. A crosslinking reaction was conducted in the same manner as inExample 1, and K_(av) was calculated. K_(av) value for the markermolecular weight of 12400 was 0.518, K_(av) value for the markermolecular weight of 67000 was 0.356, K_(av) value for the markermolecular weight of 115000 was 0.311, and K_(av) value for the markermolecular weight of 440000 was 0.152. The exclusion limit molecularweight calculated from these K_(av) values was 2.1×10⁶. The K_(av) valueand the exclusion limit molecular weight of the obtained beads weresmaller than the K_(av) value and the exclusion limit molecular weightof the beads obtained in the conditions of Example 1 to Example 4.

Evaluation Method of First Embodiment

<Crosslinking of Cellulose Beads>

40 mL of porous cellulose beads obtained in each Example was moved to areaction vessel, and 24.4 mL of a 2 N NaOH aqueous solution (preparedfrom NaOH available from NACALAI TESQUE, INC., and distilled water) wasadded. The temperature was adjusted to 40° C. Then, 24.4 mg of sodiumborohydride, and 6.0 mL of Denacol EX-314 (available from Nagase ChemteXCorporation) containing glycerol polyglycidyl ether as a crosslinkingagent were added, and stirred at 40° C. for 5 hours. After the reaction,the beads were washed with distilled water of 20 times or more of thevolume of the beads under suction filtration to obtain crosslinkedcellulose beads.

<Measurement of Gel Distribution Coefficient (K_(av))>

(1) Column Packing

The porous cellulose beads were dispersed in RO water and degassed for 1hour. The degassed porous cellulose beads or adsorbent were packed in acolumn (Tricorn 10/300 available from GE Healthcare Japan) at a linearvelocity of 105 cm/h. Thereafter, the eluent at pH 7.5 (129 mL) wasallowed to run through the column at linear velocity of 26 cm/h.

(2) Addition of Marker

As the marker, those listed below were used.

-   -   Blue Dextran 2000 (available from Pharmacia Fine Chemicals)    -   Cytochrome C (available from Wako Pure Chemical Industries,        Ltd.), molecular weight 12400    -   Bovine Serum Albumin (available from Wako Pure Chemical        Industries, Ltd.), molecular weight 67000    -   IgG derived from human (available from SIGMA), molecular weight        115000    -   Ferritin (available from SIGMA), molecular weight 440000    -   Thyroglobulin (available from SIGMA), molecular weight 660000

While the eluent was allowed to run through the column at linearvelocity of 26 cm/h, each 12 μL of the aforementioned markers dilutedinto 5 mg/mL with a buffer at pH 7.5 was injected. The concentration ofthe marker was finely adjusted each time.

(3) Measurement

DGU-20A3, SCL-10A, SPD-10A, LC-10AD, SIL-20AC and CTO-10AC (eachavailable from SHIMADZU Corporation) were used as measuring instruments,and LCsolution was used as measurement software. 50 mL graduatedcylinder was used for measuring the liquid amount.

UV monitoring and measurement of the liquid amount started at the sametime with injection of markers.

1) The liquid amount corresponding to the first peak of blue dextran wasmade as V₀ (mL).

2) The liquid amount corresponding to the peak of each marker was madeas V_(R) (mL).

3) The total volume of the porous cellulose beads or the adsorbent inthe column was made as V_(t) (mL).

4) Calculation

The distribution coefficient (K_(av)) of each marker was calculated byfollowing formula.K _(av)=(V _(R) −V ₀)/(V _(t) −V ₀)

5) Calculation of the Maximum Pore Size

K_(av) of each marker and the logarithm of the molecular weight wereplotted, and the slope and the intercept of following formula weredetermined from the part showing linearity.K _(av) =k×L _(n)(molecular weight)+b

Then, the molecular weight when K_(av) was 0, namely, the exclusionlimit molecular weight was determined from the determined slope andintercept.

2. Examples Regarding Improvement in Carrier for Ligand Immobilization(Second Embodiment)

Next, examples regarding the improvement in the carrier for ligandimmobilization (second embodiment) is described. The physical propertiesof the cellulose porous beads, the crosslinked beads (hereinafter, thesealso collectively referred to as sample beads), and the adsorbentobtained by binding a ligand to the crosslinked beads, used or obtainedin the following examples regarding the second embodiment weredetermined in the following manner.

I: Exclusion Limit Molecular Weight

(1) Column Packing Operation

The sample beads were dispersed in RO water (reverse osmosis membranepurified water) and degassed for 1 hour. The degassed sample beads werepacked in a column (Tricorn 10/300 available from GE Healthcare Japan)at a linear velocity of 105 cm/h. Thereafter, eluent at pH 7.5 (129 mL)was allowed to run through the column at linear velocity of 26 cm/h.

(2) Markers

-   -   Blue Dextran 2000 (available from Pharmacia Fine Chemicals)    -   Thyroglobulin (available from SIGMA), MW 660,000    -   Ferritin (available from SIGMA), MW 440,000    -   IgG derived from human (available from SIGMA), MW 115,000    -   Bovine Serum Albumin (available from Wako Pure Chemical        Industries, Ltd.), MW 67,000    -   Cytochrome C (available from Wako Pure Chemical Industries,        Ltd.), MW 12,400    -   Bacitracin (available from Wako Pure Chemical Industries, Ltd.),        MW 1,400

(3) Measuring Instruments and Software

Names of instruments: DGU-20A3, SCL-10A, SPD-10A, LC-10AD, SIL-20AC,CTO-10AC (each available from SHIMADZU Corporation)

Name of software: LCsolution

(4) Measurement

While the eluent was allowed to run through the column at a linearvelocity of 26 cm/h, each 12 μL of the aforementioned markers dilutedinto 5 mg/mL with a buffer at pH 7.5 was injected. The concentration ofthe marker was finely adjusted each time. UV monitoring and measurementof the liquid amount started at the same time with injection of markers.

a) The liquid amount corresponding to the first peak of blue dextran wasmade as V₀ (mL).

b) The liquid amount corresponding to the peak of each marker was madeas V_(R) (mL).

c) The total volume of the sample beads in the column was made as V_(t)(mL).

(5) Calculation

The gel phase distribution coefficient (K_(av)) of each marker wascalculated according to the following formula.K _(av)=(V _(R) −V ₀)/(V _(t) −V ₀)

K_(av) of each marker and the logarithm of the molecular weight wereplotted, and the slope and the intercept of following formula weredetermined from the part showing linearity.K _(av) =k×L _(n)(molecular weight)+bThen, the molecular weight at K_(av) of 0 was determined from thedetermined slope and intercept, and the result was taken as theexclusion limit molecular weight.

II: Shrinkage Rate at Shrinking and Crosslinking Reaction

Using the total amounts of the cellulose porous beads before and afterthe shrinking and crosslinking reaction, the sum total of the gel volumewas calculated in the following method. The gel volume V₁ of thepolysaccharide porous beads before shrinking and crosslinking of thepolysaccharide porous beads, and the gel volume V₂ of the polysaccharideporous beads after shrinking and crosslinking of the polysaccharideporous beads were determined.

(Measurement Method of Gel Volume)

The reaction solution before shrinking and crosslinking reaction orafter shrinking and crosslinking reaction was washed, and replaced withRO water, and thus a sample slurry was prepared (the gel volumeconcentration approximately 30-70% by volume). The slurry was added to a50 mL centrifugal tube, and the centrifugal tube was fixed on a smallvibrator (VIBRATORY PACKER, VP-4D available from SINFONIA TECHNOLOGY),and vibration was applied at temperature 25° C. until the beads volumeno longer changed. Then, the gel volumes V₁, V₂ were measured fromgraduations on the centrifugal tube, and the shrinkage rate wasdetermined according to the above-described calculation formula.

III: 5% Dynamic Binding Capacity

(1) Preparation of Solution

The following solutions from A to E and neutralization solution wereprepared, and they were defoamed before being used.

Solution A: PBS buffer at pH 7.4 was prepared by using “Phosphatebuffered saline” available from Sigma and RO water (reverse osmosismembrane purified water).

Solution B: 35 mM sodium acetate aqueous solution at pH 3.5 was preparedby using acetic acid, sodium acetate and RO water.

Solution C: 1 M acetic acid aqueous solution was prepared by usingacetic acid and RO water.

Solution D: An IgG aqueous solution at concentration of 1 mg/mL wasprepared by using Gammagard (polyclonal antibody) available from Baxter,and the aforementioned solution A.

Solution E: 6 M urea aqueous solution was prepared by using urea and ROwater.

Neutralization solution: 2 M tris(hydroxymethyl)aminomethane wasprepared by using tris(hydroxymethyl)aminomethane and RO water.

(2) Packing, Preparation

AKTA explorer 100 (available from GE Healthcare) was used as anapparatus for column chromatography. 3 mL of an adsorbent sample (inwhich a ligand is bound to crosslinked beads) was added in a columnhaving diameter of 0.5 cm and a height of 15 cm and packed by allowing0.2 M NaCl aqueous solution (in RO water) to run through the column at alinear velocity of 230 cm/h for 15 minutes. 15 mL sampling tubes wereset on a fraction collector, and tubes for sampling eluates werepreliminarily charged with neutralization solution.

(3) Purification of IgG

15 mL of solution A was allowed to run through the column and then 150mL of solution D was allowed to run though. Then, after allowing 21 mLof solution A to run through, 12 mL of solution B was allowed to runthrough to elute IgG. Then, 6 mL of solution C, 6 mL of solution E, and15 mL of solution A were allowed to run through. The flow rate of eachliquid was 1 mL/min so that the contact time with the adsorbent was 3minutes.

(4) Dynamic Binding Capacity

The dynamic binding capacity (5% DBC) of IgG was determined from the IgGamount adsorbed to the adsorbent before 5% breakthrough of IgG and thevolume of the adsorbent.

IV: 20% Compressive Stress

(1) Preparation of Sample

Pure water was added to sample beads to prepare a slurry (concentrationabout 50% by volume). Homogenizing and defoaming operation consisting ofhomogenization by stirring of the slurry, and the subsequent defoamingunder reduced pressure for not less than 30 minutes was repeated forthree times to obtain efoamed slurry. Separately from this operation,the homogenizing and defoaming operation was conducted for not less than90 minutes while the object to be treated was changed to pure water toobtain defoamed water.

(2) Preparation of Beads Packing Syringe

A disposable filter (pore diameter 5.00 μm, hydrophilic) was attached tothe tip end of disposer syringe of 5 mL with a lure lock (trade name:NORM-JECT) available from HANKE SASS WOLF. A piston of the syringe wasremoved, and about 3 mL of defoamed water was introduced from the rearend side of the syringe. Before the defoamed water falls below themarked line of 0 mL, defoamed slurry was introduced. An aspirator wasconnected on the secondary side of the disposable filter, and thedefoamed slurry was aspirated with care so that the liquid level did notfall below the beads level. Aspiration was stopped when the liquid levellowered to about 0.5 mL above the beads level. The subsequent operationswere conducted while the defoamed water was appropriately added so thatthe liquid level did not fall below the beads level. The defoamed slurrywas added or beads were removed under vibration and the beads level wasadjusted to the marked line of 3 mL, and it was checked that the beadslevel did not lower even when vibration was applied. Defoamed water wasadded slowly until the defoamed water spilled over in such a manner thatthe beads did not swirl around, and a piston was inserted with care sothat foams were not included (beads packing syringe).

(3) Measurement

A 10K load cell was attached to a FUDOH RHEO METER available fromRHEOTECH, and the displacement rate was set at 2 cm/MIN of the dial, andthe beads packing syringe was set, and displacement of the pistonstarted. The relationship between the displacement and the stress wasrecorded, and 20% compressive stress was determined according to thefollowing formula.20% compressive stress=stress when packing beads are compressed by 20%stress directly before piston reaches beads level

V: Linear Velocity of Critical Compression

(1) Column Packing Operation

Sample beads (89.5 mL) were dispersed in RO water, and packed in acolumn (available from MILLIPORE, inner diameter 2.2 cm) at a linearvelocity of 300 cm/h.

(2) Measurement

The column was loaded on AKTApilot (available from GE Healthcare), andRO water was allowed to run though at flow rate of 5 mL/min (linearvelocity 79 cm/hr). Subsequently, the flow rate was increased by 5mL/min stepwise. Linear velocity of critical compression was defined asthe linear velocity at the time when the column inlet pressurecontinuously increases and the liquid could not run through (in thistest, at the time when the inlet pressure exceeded 2 MPa).

Production Example 1

8.65 g of powdery cellulose (pharmacopoeia cellulose “PH-F20JP”available from Asahi Kasei Chemicals Corporation) was dispersed in 112 gof water, and retained at 4° C. Then, 35 g of 34.5 wt % sodium hydroxideaqueous solution was added and stirred. After stirring for 20 minutes,16 g of water was added so that the concentration of the sodiumhydroxide was 7.0 wt % and the temperature was raised to 15° C. Thetemperature of 890 g of 1,2-dichlorobenzene solution containing 9.2 g ofsorbitan monooleate was kept at 15° C., and the cellulose microdispersion obtained above was dispersed in this solution. The dispersionwas put into a cylindrical container having an inner diameter of 85 mm(hereinafter, referred to as a first container) equipped with atwo-stage turbine blade having a blade diameter of 45 mm and an intervalof blades of 75 mm, and the dispersion was stirred at a speed of 600 rpmfor 10 minutes. Then, 150 mL of methanol was added to obtain cellulosein the form of beads. The obtained cellulose beads were washed withmethanol, and then washed with water. The obtained cellulose beads wereclassified by using a mesh with an opening of sieve of 38 μm and a meshwith an opening of sieve of 90 μm, and cellulose particles within therange of 38 μm to 90 μm were collected.

The obtained cellulose porous beads were replaced with2-methyl-2-propanol, and freeze-dried, and then analyzed by a scanningelectron microscope (S-800 available from Hitachi, Ltd., hereinafterreferred to as SEM), and it was confirmed that the beads were porousbeads. The exclusion limit molecular weight calculated from the geldistribution coefficient (K_(av)) of the obtained beads was 6.0×10¹⁰.

Example 6

(1) Shrinking and Crosslinking Step

100 mL of gel of cellulose porous beads (water-washed product) obtainedin Production example 1 was placed on a glass filter, and solventreplacement operation of repulping with ethanol, and removing theethanol by aspiration was repeated for four times. The amount of ethanolwas 233 mL for the first to third times of the solvent replacementoperation, and 167 mL for the fourth time of the solvent replacementoperation. After the solvent replacement operation, the beads were movedto a cylindrical container having an inner diameter of 85 mm, equippedwith a paddle blade of 39 mm in blade diameter (hereinafter, referred toas a second container), and the total volume was adjusted to 149 mL byadding the same solvent, and then the temperature was raised to 40° C.Further, 80 mL of epichlorohydrin was added, and stirred at a number ofrevolutions of 200 rpm for 30 minutes. Then, a mixture consisting of 10mL of 17 M NaOH aqueous solution and 86 mL of water was added, andstirred at a number of revolutions of 350 rpm for one hour and 30minutes, and thus cellulose porous beads were shrunk and crosslinked(the reaction refers to a shrinking and crosslinking main reaction).Epichlorohydrin concentration in the shrinking and crosslinking mainreaction solution was 24.6% by volume, the organic solvent proportion(ethanol rate) was 0.61, NaOH concentration was 0.70 M, and celluloseporous beads concentration (slurry concentration) was 30.8% by volume.After conducting an additional treatment of adding 9.6 mL of 17 M NaOHaqueous solution and stirring at a number of revolutions of 350 rpm for1.5 hours three times, the reaction solution was filtered, and theresidue was washed with 20% ethanol water, followed by water to obtainintermediate crosslinked beads. The shrinkage rate by the shrinking andcrosslinking step was determined, and shown in the following Table 1.

(2) Additional Crosslinking Step

Water was added to the whole of the obtained intermediate crosslinkedbeads to adjust the entire volume to 117 mL, and moved to the secondcontainer used in the shrinking and crosslinking step, and then thetemperature was raised to 40° C. Then, 38 g of sodium sulfate was added,and stirred at a number of revolutions of 150 rpm for 10 minutes, andthen 33 mL of epichlorohydrin was added, and stirred at a number ofrevolutions of 250 rpm for 10 minutes. Then, 21 mL of 17 M NaOH aqueoussolution was added, and stirred at a number of revolutions of 300 rpmfor 2.5 hours, and eventually 5.1 mL of 17 M NaOH aqueous solution wasadded and further stirred for 2.5 hours. The reaction solution wasfiltered, and the residue was washed with water to obtain crosslinkedbeads. The whole of the obtained crosslinked beads were put into a glassErlenmeyer flask, and diluted with RO water so that the total amount was200 mL. Then, the opening was lidded with two sheets of aluminum foil,and the flask was heated at 127° C. for 60 minutes in an autoclave, andthus the remaining epoxy groups were substituted by glyceryl groups.After allowing to cool to room temperature, the beads were washed on aglass filter with 200 mL of RO water. The beads after autoclaving wereclassified by using a mesh with an opening of sieve of 38 μm and a meshwith an opening of sieve of 90 μm to collect crosslinked beads withinthe range of 38 μm to 90 μm.

(3) Preparing Step of Protein A

Referring to WO2012/133349, as orientation-controlled protein A, aconnected body of five modified-C domains as described in WO2012/133349was prepared.

(4) Ligand Immobilizing Step

3.5 mL of the crosslinked beads obtained in the additional crosslinkingstep were introduced in a centrifugal tube, and RO water was added sothat the total amount was 6 mL. The centrifugal tube was attached on amixing rotor (MIX ROTOR MR-3 available from AS ONE Corporation) at 25°C., and stirred. Next, sodium periodate was dissolved in RO water, and2.0 mL of 11.16 mg/mL of sodium periodate aqueous solution was added andstirred at 25° C. for 1 hour. Following the reaction, the beads werewashed with RO water on a glass filter (11GP100 available from SHIBATACO., LTD.) until the electric conductivity of the filtrate was not morethan 1 μS/cm to obtain formyl group-containing crosslinked beads. Theelectric conductivity of the washing filtrate was measured by aconductivity meter (ECTester10 Pure+available from EUTECH INSTRUMENTS).

On a glass filter (11GP100 available from SHIBATA CO., LTD.), 3.5 mL ofthe obtained formyl group-containing crosslinked porous cellulose beadswere replaced with 0.6 M citrate buffer (in RO water) at pH 12. Using0.6 M citrate buffer at pH 12, the formyl group-containing crosslinkedporous cellulose beads after replacement were put into a centrifugaltube, and the liquid amount was adjusted so that the total volume was7.5 mL. 0.98 g of an aqueous solution containing orientation-controlledprotein A obtained in the protein A preparing step (concentration ofprotein A is 53.8 mg/mL) was added thereto, and then allowed to reactunder stirring at 6° C. for 23 hours by using a MIX ROTOR (MIX ROTORMR-3 available from AS ONE Corporation).

Thereafter, the reaction solution was collected (reaction solution 1),and replaced with 0.1 M sodium citrate aqueous solution (in RO water) atpH 8, and stirred at 6° C. for 4 hours by using a MIX ROTOR (MIX ROTORMR-3 available from AS ONE Corporation). Subsequently, 1.93 mL of adimethylamine borane aqueous solution (in RO water) in a concentrationof 5.5% by mass was added and stirred at 6° C. for 1 hour. Then, thereaction temperature was raised to 25° C., and allowed to react at 25°C. for 18 hours under stirring by using a MIX ROTOR (MIX ROTOR MR-3available from AS ONE Corporation). After reaction, the reactionsolution was collected (reaction solution 2). UV absorbance of themaximum absorbance around 278 nm of the reaction solutions 1 and 2 wasmeasured, and the measured value was subtracted from the loaded ligandamount to calculate the immobilized amount of protein A. The result isshown in Table 1.

The beads after reaction was washed with RO water in an amount of threetimes the volume of the beads on the glass filter (11GP100 availablefrom SHIBATA CO., LTD.). Subsequently, 0.1 N citrate aqueous solution(in RO water) in an amount of three times the volume was added, and 0.1N citric acid aqueous solution (in RO water) was added to the beads sothat the total amount was not less than 30 mL, and these were put into acentrifugal tube, and thus acid washing was conducted at 25° C. for 30minutes under stirring.

After acid washing, the beads were washed with RO water in an amount ofthree times the volume of the beads on the glass filter (11GP100available from SHIBATA CO., LTD.), and then an aqueous solution (in ROwater) containing 0.05 M sodium hydroxide and 1 M sodium sulfate in anamount of three times the volume was added. Then, aqueous solutioncontaining 0.05 M sodium hydroxide and 1 M sodium sulfate was added sothat the total amount was not less than 30 mL, and these were put into acentrifugal tube, and thus alkali washing was conducted at roomtemperature for 30 minutes under stirring.

After alkali washing, the beads were washed with RO water in an amountof 20 times the volume of the beads on the glass filter (11GP100available from SHIBATA CO., LTD.), and then 0.1 N sodium citrate aqueoussolution (in RO water) in an amount of three times the beads was added.After confirming that the filtrate was neutral, the beads were washedwith RO water until the electric conductivity of the washing filtratewas not more than 1 μS/cm to obtain an adsorbent in which the objectiveorientation-controlled protein A was immobilized. The electricconductivity of the washing filtrate was measured by a conductivitymeter (ECTester10 Pure+available from EUTECH INSTRUMENTS).

The linear velocity of critical compression and the 20% compressivestress of the crosslinked beads (after the additional crosslinking step)obtained in the manner as described above, and the 5% dynamic bindingcapacity (contact time 3 minutes) of the adsorbent were measured. Thelinear velocity of critical compression of the crosslinked beads was1658 cm/hr. The remaining results are shown in Table 1.

Examples 7-8

The same operation as in Example 6 was conducted except that ethanolused in the shrinking and crosslinking step was changed to each organicsolvent shown in Table 1, and the organic solvent proportion, theepichlorohydrin concentration, the NaOH concentration, and the slurryconcentration (cellulose porous beads concentration) in the shrinkingand crosslinking step were changed to the values shown in Table 1. Theresults are shown in Table 1.

TABLE 1 Example 6 Example 7 Example 8 Organic solvent*¹ Ethanol EthanolDMSO Concentration of 24.6 12.3 12.3 epichlorohydrin (volume %) Organicsolvent proportion 0.61 0.59 0.53 Concentration of NaOH 0.70 0.60 0.60(M) Concentration of slurry 30.8 30.8 30.8 (volume %) Shrinkage rate atshrinking 36.7 22.4 26.8 and crosslinking step (%) 20% compressivestress 0.112 0.072 0.096 (MPa) Immobilized amount of 13.4 12.6 12.8protein A (mg/mL - gel) 5% dynamic binding capacity 41.7 44.4 43.7(mg/mL - gel) *¹DMSO = Dimethyl sulfoxide

Examples 9-10, Comparative Example 3

(1) Shrinking and Crosslinking Step

On a glass filter, 15 mL of gel of cellulose porous beads (water washedproduct) obtained in Production example 1 was placed, and a solventreplacement operation of repulping with 15 mL of a specific solventshown in Table 2, and removing the specific solvent by aspiration wasrepeated three times. After the solvent replacement operation, the wholeof the gel was put into a centrifugal tube, and the volume was adjustedso that the total amount was 17.5 mL by adding the same specificsolvent, and further 8.7 mL of epichlorohydrin was added and stirredovernight. Subsequently, 10.3 mL of water and 2.1 mL of 17 M NaOHaqueous solution were added, and stirred at temperature of 40° C. forone hour and 30 minutes to cause shrinking and crosslinking of thecellulose porous beads (shrinking and crosslinking main reaction). Theepichlorohydrin concentration, the organic solvent proportion, the NaOHconcentration and the cellulose porous beads concentration (slurryconcentration) in the shrinking and crosslinking main reaction solutionare shown in Table 2 below. Then, 1.05 mL of 17 M NaOH aqueous solutionwas added, and stirred for 1.5 hour, and then additional treatment ofadding 1.05 mL of 17 M NaOH aqueous solution and stirring for 2 hourswas conducted, and then they were filtered, and the residue was washedwith 20% specific solvent aqueous solution, followed by water to obtainintermediate crosslinked beads. The shrinkage rate by the shrinking andcrosslinking step was determined, and shown in Table 2 below.

(2) Additional Crosslinking Step

The whole of the obtained intermediate crosslinked beads were put into acentrifugal tube, and the entire volume was adjusted to 17.5 mL byaddition of water, and then the temperature was raised to 40° C. Then,5.67 g of sodium sulfate, 4.95 mL of epichlorohydrin, and 3.14 mL of 17M NaOH aqueous solution were added, and stirred at temperature 40° C.for 2.5 hours, and finally 0.76 mL of 17 M NaOH aqueous solution wasadded and stirred for another 2.5 hours. The reactant was filtered, andthe residue was washed with water to obtain crosslinked beads. The wholeof the obtained crosslinked beads were put into a glass Erlenmeyerflask, and diluted with RO water so that the total volume was 50 mL, andthen the opening was lidded with two sheets of aluminum foil, and theflask was heated at 127° C. for 60 minutes in an autoclave. Afterallowing to cool to room temperature, the beads were washed with 50 mLof RO water on a glass filter, to substitute the remaining epoxy groupswith glyceryl groups. The beads after autoclaving were classified byusing a mesh with an opening of sieve of 38 μm and a mesh with anopening of sieve of 90 μm, and an adsorbent within the range of 38 μm to90 μm was collected.

(3) Preparing Step of Protein A

Referring to Examples of WO2011/118699, as modified protein A, aconnected body of five modified-C domains having alkali resistance asdescribed in WO2011/118699 was prepared.

(4) Ligand Immobilizing Step

5 mL of crosslinked porous cellulose beads obtained in the additionalcrosslinking step were introduced in a centrifugal tube, and RO waterwas added so that the total amount was 7.5 mL. The centrifugal tube wasattached on a mixing rotor (MIX ROTOR MR-3 available from AS ONECorporation) at 25° C. and stirred. Next, sodium periodate was dissolvedin RO water, and 2.5 mL of 12.84 mg/mL of sodium periodate aqueoussolution was added and stirred at 25° C. for 1 hour. After the reaction,the beads were washed with RO water on a glass filter (11GP100 availablefrom SHIBATA CO., LTD.) until the electric conductivity of the filtratewas not more than 1 μS/cm to obtain formyl group-containing crosslinkedporous cellulose beads. The electric conductivity of the washingfiltrate was measured by a conductivity meter (ECTester10 Pure+availablefrom EUTECH INSTRUMENTS).

5 mL of the obtained formyl group-containing crosslinked porouscellulose beads were replaced with 0.25 M citrate buffer (prepared byusing trisodium citrate dihydrate and RO water) on a glass filter(11GP100 available from SHIBATA CO., LTD.). Using 0.25 M citrate buffer,the formyl group-containing crosslinked porous cellulose beads afterreplacement were put into a centrifugal tube, and the liquid amount wasadjusted so that the total volume was 7.5 mL. 2.13 g of aqueous solutioncontaining alkali resistant protein A obtained in the protein Apreparing step (protein A concentration 70.3 mg/mL) was added thereto,and pH was adjusted to 12 with 0.08 N sodium hydroxide aqueous solution,and then allowed to react under stirring at 6° C. for 23 hours by usinga MIX ROTOR (MIX ROTOR MR-3 available from AS ONE Corporation).

Thereafter, 2.4 M citric acid aqueous solution (prepared by using citricacid monohydrate and RO water) was added until pH of the reactionsolution was 5, and stirred at 6° C. for 4 hours by using a MIX ROTOR(MIX ROTOR MR-3 available from AS ONE Corporation). Subsequently, 1.13mL of a dimethylamine borane aqueous solution (in RO water) inconcentration of 5.5% by mass was added and stirred at 6° C. for 1 hour,and then the reaction temperature was raised to 25° C., and allowed toreact at 25° C. for 18 hours under stirring. After reaction, thereaction solution was collected, and UV absorbance of the maximumabsorbance around 278 nm was measured, and the measured value wassubtracted from the loaded ligand amount to calculate the immobilizedamount of protein A.

In the subsequent steps, the beads after reaction were washed (acidwashing, alkali washing, neutralization) by repeating the sameoperations as in the ligand immobilizing step in Example 6, and thus anadsorbent in which objective alkali resistant protein A was immobilizedwas obtained.

20% compressive stress of the crosslinked beads (after the additionalcrosslinking step) obtained in the manner as described above, and theamount of immobilized protein A per 1 mL of the adsorbent, and the 5%dynamic binding capacity (contact time 3 minutes) of the adsorbent weremeasured. The results are shown in Table 2.

TABLE 2 Comparative Example 3 Example 9 Example 10 Specific solventWater Mixed solvent*¹ Ethanol Concentration of 22.5 22.5 22.5epichlorohydrin (volume %) Organic solvent proportion 0 0.59 0.59Concentration of NaOH 1.19 1.19 1.19 (M) Concentration of slurry 38.938.9 38.9 (volume %) Shrinkage rate at shrinking −5 16 37 andcrosslinking step (%) 20% compressive stress 0.048 0.092 0.14 (MPa)Immobilized amount of 15.0 15.9 12.7 protein A (mg/mL - gel) 5% dynamicbinding 40.1 42.5 42.6 capacity (mg/mL - gel) *²Mixed solvent ofmethanol/dimethyl sulfoxide = 1/1 (volume ratio)

Examples 11-17

The same operation as in Examples 9-10 was conducted except that thesolvent used in the shrinking and crosslinking step was changed to thoseshown in Table 3. The results are shown in Table 3. The relationshipbetween the shrinkage rate and the 20% compressive stress is shown inFIG. 5.

TABLE 3 Example 11 Example 12 Example 13 Example 14 Example 15 Example16 Example 17 Specific solvent*¹ Methanol Acetonitrile IPA DMSO DMAAcetone Dioxane Concentration of 22.5 22.5 22.5 22.5 22.5 22.5 22.5epichlorohydrin (volume %) Organic solvent 0.59 0.59 0.59 0.59 0.59 0.590.59 proportion Concentration of 1.19 1.19 1.19 1.19 1.19 1.19 1.19 NaOH(M) Concentration of 38.9 38.9 38.9 38.9 38.9 38.9 38.9 slurry (volume%) Shrinkage rate at 10 14 40 43 60 23 23 shrinking and crosslinkingstep (%) 20% compressive 0.072 0.072 0.124 0.18 0.272 0.092 0.096 stress(MPa) *¹IPA = Isopropanol, DMSO = Dimethyl sulfoxide, DMA = Dimethylacetamide

Comparative Example 4

(1) First Crosslinking Step

20 mL of gel of the cellulose porous beads (water washed product)obtained in Production example 1 was placed on a glass filter, andrepulped with RO water, and then RO water was removed by aspiration. Thewhole of the beads were put into a centrifugal tube, and 12.2 mL of 2 MNaOH aqueous solution was added and mixed. Further 6.6 mL of glycerolpolyglycidyl ether (Denacol EX314 available from Nagase ChemteXCorporation) and 7.6 g of sodium sulfate were added, and stirred attemperature of 40° C. for 5 hours to cause crosslinking of the celluloseporous beads. Then, these were filtered, and the residue was washed withwater to obtain intermediate crosslinked beads.

(2) Second Crosslinking Step

The whole of the obtained intermediate crosslinked beads were put into acentrifugal tube, and 12.2 mL of 2 M NaOH aqueous solution was added andmixed. Further, 6.6 mL of glycerol polyglycidyl ether and 7.6 g ofsodium sulfate were added, and stirred at temperature of 40° C. for 5hours, and then filtered, and the residue was washed with water toobtain crosslinked beads. The whole of the obtained crosslinked beadswere put into a glass Erlenmeyer flask, and diluted with RO water sothat the total amount was 50 mL, and then the opening was lidded withtwo sheets of aluminum foil, and the flask was heated at 127° C. for 60minutes in an autoclave. After allowing to cool to room temperature, thebeads were washed with 50 mL of RO water on a glass filter to substitutethe remaining epoxy groups with glyceryl groups. The beads afterautoclaving were classified by using a mesh with an opening of sieve of38 μm and a mesh with an opening of sieve of 90 μm, and crosslinkedbeads within the range of 38 μm to 90 μm was collected. The shrinkagerate after crosslinking was 0%.

In the subsequent steps, (4) ligand immobilizing step was conducted inthe same manner as in Examples 9-10, and thus an adsorbent in whichobjective alkali resistant protein A being immobilized was obtained.

20% compressive stress of the crosslinked beads (after the secondcrosslinking step) obtained in the manner as described above, and theamount of immobilized protein A per 1 mL of the adsorbent, and the 5%dynamic binding capacity (contact time 3 minutes) of the adsorbent weremeasured. The 20% compressive stress was 0.084 MPa, the immobilizedamount was 14.8 mg/mL-gel, and the 5% dynamic binding capacity (DBC) was22.0 mg/mL-gel.

INDUSTRIAL APPLICABILITY

The porous cellulose beads according to the first embodiment can be usedas an adsorbent for various substances by addition of varioussubstituents. The carrier for ligand immobilization according to thesecond embodiment can be turned into an adsorbent by immobilizing aligand.

The invention claimed is:
 1. A carrier for ligand immobilization,obtained by a process comprising shrinking polysaccharide porous beadssuch that a shrinkage rate is not less than 10%, and crosslinking thepolysaccharide porous beads, whereinShrinkage rate (%)=(1−V ₂ /V ₁)×100 wherein V₁ represents a gel volumeof the polysaccharide porous beads before the shrinking, and V₂represents a gel volume of the polysaccharide porous beads after theshrinking.
 2. The carrier according to claim 1, wherein the shrinkingcomprises contacting the polysaccharide porous beads into contact with awater-soluble organic solvent and alkali water.
 3. The carrier accordingto claim 2, wherein the shrinking is carried out in the presence of acrosslinking agent such that the polysaccharide porous beads arecrosslinked during the shrinking, or the crosslinking is carried outafter the shrinking such that shrunk polysaccharide porous beadsobtained by the shrinking are crosslinked.
 4. The carrier according toclaim 3, wherein the process further comprises, after the crosslinking,additionally contacting the polysaccharide porous beads with acrosslinking agent and alkali water at least one time.
 5. The carrieraccording to claim 1, wherein the polysaccharide porous beads comprise apolysaccharide which is cellulose or agarose.
 6. The carrier accordingto claim 2, wherein the water-soluble organic solvent is at least oneselected from the group consisting of an alcohol solvent, a sulfoxidesolvent, an amide solvent, a ketone solvent, and an ether solvent. 7.The carrier according to claim 4, wherein an alcohol solvent is not usedin the additional contacting crosslinking.
 8. An adsorbent, obtained byimmobilizing a ligand on the carrier according to claim
 1. 9. Theadsorbent according to claim 8, wherein the ligand is an affinityligand.
 10. The adsorbent according to claim 9, wherein the affinityligand is protein A, protein G, or protein L.
 11. A method for purifyingan antibody by affinity chromatography, the method comprising:contacting a source material with the adsorbent according to claim 9,such that an antibody is adsorbed on the adsorbent; washing the antibodyadsorbed on the adsorbent; adding an eluent such that the antibody isliberated from the adsorbent; and collecting the liberated antibody fromthe eluent.
 12. The carrier according to claim 1, wherein thepolysaccharide porous beads have an exclusion limit molecular weight of1.0×10⁵ to 1.0×10¹².
 13. The carrier according to claim 1, wherein thepolysaccharide porous beads have an exclusion limit molecular weight of1.0×10⁶ to 1.0×10¹¹.
 14. The carrier according to claim 2, wherein thewater-soluble organic solvent is at least one selected from the groupconsisting of methanol, ethanol, propanol, dimethyl sulfoxide, dimethylformamide, dimethyl acetamide, N-methylpyrrolidone, acetone, dioxane,and tetrahydrofuran.
 15. The carrier according to claim 1, wherein theprocess comprises shrinking the polysaccharide porous beads such thatthe shrinkage rate is 10% to 60%.
 16. The carrier according to claim 1,wherein the process comprises shrinking the polysaccharide porous beadssuch that the shrinkage rate is 20% to 50%.
 17. The carrier according toclaim 1, wherein the carrier has a 20% compressive stress of 0.06 MPa to0.28 MPa.
 18. The carrier according to claim 1, wherein the carrier hasa 20% compressive stress of 0.072 MPa to 0.20 MPa.
 19. The carrieraccording to claim 1, wherein the carrier has a 20% compressive stressof 0.092 MPa to 0.20 MPa.
 20. The carrier according to claim 1, whereinthe carrier has a particle diameter of 10 μm to 200 μm.